Klebsiella pneumoniae (KP) and Pseudomonas aeruginosa (PA) are Gram Negative Bacteria (GNB) that are among the most prevalent and virulent pathogens associated with wound infections in combat personnel. They can cause serious clinical syndromes including abscess formation, cellulitis, disseminated infection, and bacteremia leading to progressive amputation, permanent impairment and death by septic shock. The growing proportion of Klebsiella pneumoniae and PA that are multi-drug resistant (MDR) complicates treatment Immunoprophylactic measures against PA and Klebsiella pneumoniae can be effective irrespective of the antibiotic resistance phenotypes.
Klebsiella pneumoniae can express two virulence-associated polysaccharides (PS): a secreted cell-associated capsular polysaccharide (CPS) that coats the bacterium and a lipopolysaccharide (LPS) that forms the outer leaflet of the outer-membrane. The polysaccharide portion of Klebsiella pneumoniae LPS is comprised of a genus-specific conserved core and a serotype specific polymer of O polysaccharide (OPS; FIG. 1) for which there are ˜9 recognized serotypes (Vinogradov E, J Biol Chem. 2002; 277(28):25070-25081; Vinogradov E, Carbohydr Res. 2001; 335(4):291-296).
Importantly, the prevalence of OPS types among clinical isolates is highly restricted. Hospital based surveys of invasive infections have revealed that four OPS serotypes (O1, O2a, O3 and O5) account for 60-80% strains causing infections in the USA and worldwide. By comparison, there are at least 80 identified CPS serotypes of which greater than 25 are associated with invasive infections in humans in the USA (Podschun R, Clin Microbiol Rev. 1998; 11(4):589-603; Hansen D S, J Clin Microbiol. 1999; 37(1):56-62); Trautmann M, Vaccine. 2004; 22(7):818-821; Cryz S J, Jr., J Clin Microbiol. 1986; 23(4):687-690). Furthermore, the incidence and prevalence of invasive infections attributed to various CPS serotypes varies dramatically worldwide; CPS types that are prevalent in one region, can be absent entirely in others (Cryz S J, Jr., J Clin Microbiol. 1986; 23(4):687-690), including potential areas of military deployment.
Despite envelopment by CPS, evidence has accumulated that Klebsiella pneumoniae LPS is accessible to antibody. LPS expression is required for protection against the alternative pathway of the complement system (Merino S, Infect Immun. 1992; 60(6):2529-2535). Long-chain OPS polymers extend beyond the capsule surface and activate the alternative complement pathway at their distal ends, too far from the cell-surface to be functional (Tomas J M, Infect Immun. 1991; 59(6):2006-2011; Williams P, J Gen Microbiol. 1983; 129(7):2181-2191; Tomas J M, Microb Pathog. 1988; 5(2):141-147). Selective pressure for OPS expression has been documented when KP is grown in human serum and absent when serum complement is heat-inactivated or KP is grown in broth culture (Camprubi S, Microb Pathog. 1992; 13(2):145-155). LPS expression has also been associated with establishment of invasive infections in animal models (Lawlor M S, Mol Microbiol. 2005; 58(4):1054-1073; Hsieh P F, PLoS One. 2012; 7(3):e33155). Short-chain LPS is also likely to be antibody accessible, as several capsule types have been documented as permeable to antibody (Meno Y, Infect Immun. 1990; 58(5):1421-1428; Williams P, J Med Microbiol. 1988; 26(1):29-35); and LPS can become further exposed by thin and incomplete encapsulation.
Klebsiella pneumoniae CPS are important virulence factors that antagonize non-specific opsonophagocytic uptake and capsule-deficient Klebsiella pneumoniae are highly attenuated (Williams P, J Gen Microbiol. 1983; 129(7):2181-2191; Domenico P, Infect Immun. 1994; 62(10):4495-4499). However, expression of CPS inhibits binding interactions by Klebsiella pneumoniae adhesins with epithelial cells, an important early step in infection; thus it is likely that CPS expression is down-regulated in the early stages of infection (Favre-Bonte S, Infect Immun. 1999; 67(2):554-561; Hennequin C, Res Microbiol. 2007; 158(4):339-347; Schembri M A, Infect Immun. 2005; 73(8):4626-4633). Numerous studies have supported the role of antibodies towards Klebsiella pneumoniae LPS in protection against invasive KP infection with encapsulated strains. Antibodies to OPS antigen induced by active immunization with purified LPS (Tomas J M, Infect Immun. 1991; 59(6):2006-2011; Clements A, Vaccine. 2008; 26(44):5649-5653; Chhibber S, Jpn J Infect Dis. 2004; 57(4):150-155), OPS:protein conjugates (Chhibber S, Indian J Exp Biol. 2005; 43(1):40-45; Chhibber S, Vaccine. 1995; 13(2):179-184), killed whole-cells (Shimoguchi K., Kansenshogaku Zasshi. 1990; 64(12):1482-1492), acapsular mutants (Lawlor M S, Infect Immun. 2006; 74(9):5402-5407), or passive transfer with polyclonal (Clements A, Vaccine. 2008; 26(44):5649-5653) or monoclonal (Held T K, Infect Immun. 2000; 68(5):2402-2409) anti-LPS antibodies have protected against fatal Klebsiella pneumoniae pneumonic and intraperitoneal infections in rodents. Parenteral immunization with formalin inactivated whole-cell encapsulated Klebsiella pneumoniae has protected against infection with the homologous encapsulated strain, and remarkably negligible anti-CPS but robust anti-LPS antibody was detected, for which the level correlated well with protection (Shimoguchi K., Kansenshogaku Zasshi. 1990;64(12):1482-1492) Immunization with purified O1 LPS has also elicited protection against O1 strains expressing different capsule types (Tomas J M, Infect Immun. 1991; 59(6):2006-2011) Anti-Klebsiella pneumoniae O1 OPS monoclonal antibodies have demonstrated enhanced opsonophagocytosis of encapsulated strains (Held T K, Infect Immun. 2000; 68(5):2402-2409), and protected against encapsulated Klebsiella pneumoniae when given by passive transfer (Rukavina T, Infect Immun. 1997; 65(5):1754-1760). Partial protection has also been obtained by antibodies directed towards the core polysaccharide that is conserved among Klebsiella pneumoniae, the diminished protection relative to anti-OPS is likely due however to steric hindrance for accessibility of the core polysaccharide to antibody in the context of long-chain OPS (Chen W H, Innate Immun. 2008; 14(5):269-278; Mandine E, Infect Immun. 1990; 58(9):2828-2833).
Immune responses to Klebsiella pneumoniae outer membrane proteins (e.g., iron regulated proteins, porins) have also protected (Chhibber S, Vaccine. 1995; 13(2):179-184; Serushago B A, J Gen Microbiol. 1989; 135(8):2259-2268; Kurupati P, Clin Vaccine Immunol. Jan 2011; 18(1):82-88). However, evidence suggests that LPS is the superior vaccine target, as antibodies to purified OMP proteins did not protect as well as antibodies to non-encapsulated whole cell preparations that included LPS (Serushago B A, J Gen Microbiol. 1989; 135(8):2259-2268) Immunization with Klebsiella pneumoniae capsular polysaccharides have protected against burn-wound Klebsiella pneumoniae infections in animal models (Cryz S J, Jr., J Infect Dis. 1984; 150(6):817-822), and passive transfer with anti-capsule antibodies recapitulated the protection seen with active vaccination (Cryz S J, Jr., Infect Immun. 1984; 45(1):139-142). As anti-LPS antibodies are protective against intraperitoneal (IP) and pneumonic Klebsiella pneumoniae infections in mice, they are also presumed to be protective against wound infections caused by Klebsiella pneumoniae. 
Generating a CPS-based vaccine that would be effective against pathogenic Klebsiella pneumoniae strains worldwide is not easily accomplished as the manufacture and establishment of acceptable immunogenicity for all components of a ≥25 valent vaccine is a major challenge. A 24-valent Klebsiella pneumoniae CPS vaccine was shown to be immunogenic in human trials (Edelman R, Vaccine. 1994; 12(14):1288-1294). However, the levels varied dramatically among serotypes, with some inducing only poor antibody levels. Importantly, antibody levels for most Klebsiella pneumoniae CPS types plunged within the 18 months of follow-up to pre-immune levels (Edelman R, Vaccine. 1994; 12(14):1288-1294; Granstrom M, J Clin Microbiol. 1988; 26(11):2257-2261). Similar responses have been seen in humans to the capsular polysaccharides of other pathogens, and in certain instances (Pace D, Vaccine. 2009; 27 Suppl 2:B30-41; Gonzalez-Fernandez A, Vaccine. 17 2008; 26(3):292-300) progressively diminished boost responses have been noted after sequential re-immunizations due to depletion of pre-committed naive B-cells (Richmond P, J Infect Dis. 2000; 181(2):761-764). Polysaccharides are thymus-independent antigens that do not activate T-cells and hence generally generate only moderate antibody titers without immunologic memory, class-switching, or affinity maturation (Pollard A J, Nat Rev Immunol. 2009; 9(3):213-220). Furthermore, whereas some polysaccharides elicit acceptable antibody levels, other polysaccharides are not immunogenic as purified antigens. Covalent chemical linkage of bacterial polysaccharides with proteins has enhanced the magnitude, quality and duration of the induced antibody, through activation of polysaccharide-specific B-cells by protein carrier specific helper T-cells, and importantly, has generated anamnestic and booster responses. Glycoconjugate vaccines are among the most costly of all vaccine types to manufacture, however, and development of multivalent conjugate formulations with >7 components (e.g., pneumococcal CPS conjugates) have been hampered by issues of epitopic suppression and interference among individual components (Dagan R, Vaccine. 2010; 28(34):5513-5523).
Since antibodies to the OPS of Klebsiella pneumoniae are protective, and the overall number and predominance of OPS types is relatively limited, it raises the possibility that a Klebsiella pneumoniae OPS vaccine approach might be a more straightforward and feasible vaccine strategy for KP. Accordingly, there has been extensive investigation over the previous decades towards vaccine strategies targeting KP LPS. Vaccine formulations utilizing whole-cell killed organisms and purified LPS, however, are unacceptably reactogenic for humans, as they elicit severe adverse reactions including high fever and malaise.
The lipid A endotoxin portion of LPS is readily cleaved by chemical means, yielding isolated O polysaccharide (OPS) or a core oligosaccharide and an O polysaccharide (COPS)(Wang X, Subcell Biochem. 2010; 53:27-51; Simon R, Infect Immun. 2011; 79(10):4240-4249). As purified polysaccharide antigens, COPS molecules have generally proven entirely refractory to antibody production in animal models (Simon R, Infect Immun. 2011; 79(10):4240-4249; Konadu E, Infect Immun. 1996; 64(7):2709-2715; Watson D C, Infect Immun. 1992; 60(11):4679-4686). However, conjugation with carrier proteins (e.g., CRM197, flagellins, porins, tetanus toxoid [TT]) has enhanced immunogenicity (Knuf M, Vaccine. 2011; 29(31):4881-4890). COPS-based conjugate vaccines have proven efficacious in animal models for several GNB pathogens (e.g., E. coli (Cryz S J, Jr., Infect Immun. 1990; 58(2):373-377; Konadu E, Infect Immun. 1994; 62(11):5048-5054), V. cholerae, PA (Cryz S J, Jr., Infect Immun. 1986; 52(1):161-165), Salmonella (Simon R, Infect Immun. 2011; 79(10):4240-4249; Konadu E, Infect Immun. 1996; 64(7):2709-2715; Watson D C, Infect Immun. 1992; 60(11):4679-4686; Svenson S B, Infect Immun. 1979; 25(3):863-872; Micoli F, PLoS One. 2012; 7(11):e47039), Shigella (Kubler-Kielb J, Carbohydr Res. 2010; 345(11):1600-1608; Robbins J B, Proc Natl Acad Sci USA. 2009; 106(19):7974-7978; Chu C Y, Infect Immun. 1991; 59(12):4450-4458)). Importantly, COPS conjugates have been well-tolerated and immunogenic in human clinical trials (Passwell J H, Infect Immun. 2001; 69(3):1351-1357; Cohen D, Infect Immun. 1996; 64(10):4074-4077; Konadu E Y, Infect Immun. 2000; 68(3):1529-1534; Konadu E Y, J Infect Dis. 1998; 177(2):383-387; Cryz S J, Jr., J Clin Invest. 1987; 80(1):51-56; Cryz S J, Jr., J Infect Dis. 1986; 154(4):682-688) and have induced functional bactericidal antibodies (Konadu E Y, Infect Immun. 2000; 68(3):1529-1534). Some COPS conjugates have demonstrated efficacy in controlled field trials. In a large randomized double-blind efficacy trial of a Shigella sonnei COPS conjugate among military recruits in Israel, significant protection was observed, for which levels of anti-S. sonnei LPS correlated with protection (Cohen D, Lancet. 1997; 349(9046):155-159). A Pseudomonas aeruginosa COPS-based conjugate vaccine was immunogenic when administered to acute trauma patients within 72 hours of hospitalization (Campbell W N, Clin Infect Dis. 1996; 23(1):179-181).
All pathogenic Pseudomonas aeruginosa express a single polar flagellum that extends from the cell surface (FIG. 2; adapted from Dasgupta N, J Bacteriol. 2000; 182(2):357-364) to enable motility, that is comprised chiefly by polymers of either type A or B flagellin proteins (Stanislaysky E S, FEMS Microbiol Rev. 1997; 21(3):243-277). There is a single B-type flagellin form (FlaB)(Verma A et al., J Bacteriol. 1998; 180(12):3209-3217), and two A-type flagellin sub-forms (FlaA) that differ in sequence by only a few amino acids and are similarly reactive with A-type specific antibodies (Brimer C D, Montie T C, J Bacteriol. 1998; 180(12):3209-3217; Arora S K et al., J Bacteriol. 2004; 186(7):2115-2122). While there have been no rigorous surveys conducted to determine the precise prevalence of strains expressing A and B type flagellin, the distribution of A and B type flagella expressing clinical isolates reported in the literature suggests that the prevalence of the two flagella types does not differ greatly (Rosok M J et al., Infect Immun. 1990; 58(12):3819-3828; Shanks K K et al., Clin Vaccine Immunol. 2010; 17(8):1196-1202).
Pseudomonas aeruginosa flagella are well established as virulence factors and protective antigens against Pseudomonas aeruginosa infections. The requirement of flagella for Pseudomas aeruginosa pathogenicity is underscored by the dramatically reduced virulence observed for strains lacking flagella in mouse models of fatal Pseudomas aeruginosa wound and respiratory infections (Montie T C et al., Infect Immun. 1982; 38(3):1296-1298; Feldman M et al., Infect Immun. 1998; 66(1):43-51). Several roles have been noted for flagella in Pseudomonas aeruginosa pathogenesis. Flagellar mediated motility is important for biofilm development, and strains lacking functional motile flagella do not establish robust biofilms in vitro and are attenuated in vivo (Klausen M et al., Mol Microbiol. 2003; 48(6):1511-1524; O'Toole G A et al., Mol Microbiol. 1998; 30(2):295-304; Arora S K et al., Infect Immun. 2005; 73(7):4395-4398). Accordingly, highly motile strains are extremely pathogenic in a mouse model of Pseudomas aeruginosa burn infection (Craven R C et al., Can J Microbiol. 1981; 27(4):458-460). Flagella have also been found as attachment and colonization factors binding to mammalian epithelial cell glycans (Arora S K et al., Infect Immun. 1998; 66(3): 1000-1007; Arora S K et al., Infect Immun. 1996; 64(6):2130-2136; Lu W et al., J Immunol. 2006; 176(7):3890-3894). Binding to mammalian Toll-like receptor 5 (TLRS) protein by Pseudomonas flagellin activates putative protective pro-inflammatory signaling pathways, however, overt inflammation due to flagellin is likely to be detrimental to the host (B alloy V et al., J Infect Dis. 2007; 196(2):289-296; Ben Mohamed F et al., PLoS One. 2012; 7(7):e39888).
Antibodies specific for Pseudomas aeruginosa flagellins elicited by active immunization, or supplied by passive transfer have conferred robust protection in animal models against respiratory (Campodonico V L et al., Infect Immun. 2011; 79(8):3455-3464; Campodonico V L et al., Infect Immun. 2010; 78(2):746-755), peritonitis (Neville L F et al., Int J Mol Med. 2005; 16(1):165-171) or burn wound (Faezi S et al., APMIS. 2013; Barnea Y et al., Burns. 2009; 35(3):390-396; Barnea Y et al., Plast Reconstr Surg. 2006; 117 (7): 2284-2291; Holder I A et al., J Trauma. 1986; 26(2):118-122; Holder I A et al., Am J Med. 1984; 76(3A):161-167; Holder I A et al., Infect Immun. 1982; 35(1):276-280) Pseudomas aeruginosa infections. The presumed mechanism of protection by anti-Fla antibodies is arrest of motility and enhancement of opsonophagocytic killing (Stanislaysky E S, FEMS Microbiol Rev. 1997; 21(3):243-277; Doring G etal., Vaccine. 2008; 26(8):1011-1024; Faezi S etal., Burns. 2011; 37(5):865-872). Protection, including for burn wound infections, has been found as specific for either A or B type flagellins (Holder I A et al., Infect Immun. 1982; 35(1):276-280; Montie T C et al., Infect Immun. 1982; 35(1):281-288). Mice immunized with bivalent preparations of type A and B flagellins purified from Pseudomas aeruginosa were protected against fatal infection in the burn-sepsis model of Pseudomas aeruginosa infection with both subtypes of flagellin expressing strains, indicating that a broadly protective bivalent Pseudomonas aeruginosa flagellin vaccine is feasible (Holder I A et al., J Trauma. 1986; 26(2):118-122; Holder I A et al., Infect Immun. 1982; 35(1):276-280). Several groups have reported robust protection against wound infections including for MDR-Pseudomonas aeruginosa by passive transfer of anti-flagellin polyclonal sera (Faezi S et al., Burns. 2011; 37(5):865-872; Drake D et al., Can J Microbiol. 1987; 33(9):755-763), as well as monoclonal antibodies directed against type specific FlaA and FlaB epitopes (Rosok M J et al., Infect Immun. 1990; 58(12):3819-3828; Barnea Y et al., Burns. 2009; 35(3):390-396; Barnea Y et al., Plast Reconstr Surg. 2006; 117(7):2284-2291; Adawi A et al., Int J Mol Med. 2012; 30(3):455-464). In one study, passive transfer of a monoclonal anti-FlaA produced equivalent survival against PA infection in burned mice as antibiotic (imipenem) treatment (Barnea Y et al., Burns. 2009; 35(3):390-396). Pseudomas aeruginosa flagellin vaccines have also been investigated in human clinical trials, and were found to be well tolerated and immunogenic (Doring G et al., Proc Natl Acad Sci USA. 26 2007; 104(26):11020-11025; Doring G, Dorner F., Behring Inst Mitt. 1997; (98):338-344; Doring G et al., Am J Respir Crit Care Med. 1995; 151(4):983-985). A double-blind randomized Phase 3 trial in cystic fibrosis patients with a bivalent Pseudomas aeruginosa A/B flagellin vaccine revealed robust and durable antibody titers, and statistically significant protection (Doring G et al., Proc Natl Acad Sci USA. 26 2007; 104(26): 11020-11025).
Pseudomas aeruginosa flagellins are not expressed at high levels natively, and hence high yield expression systems are required to establish feasibility for large-scale production. Pseudomas aeruginosa flagellins are readily expressed and purified from heterologous Gram Negative Bacteria (GNB) expression systems, including Salmonella and Escherichia coli (Campodonico V L et al., Infect Immun. 2011; 79(8):3455-3464; Kelly-Wintenberg K et al., J Bacteriol. 1989; 171(11):6357-6362; Inaba S et al., Biopolymers. 2013; 99(1):63-72). The FliD capping protein is an essential factor for polymerization of secreted flagellin monomers into flagella polymers, and in the absence of effective FliD function, flagellin monomers are secreted into the extracellular space in an unpolymerized form. The FliD protein of E. coli is an effective substitute for Pseudomas aeruginosa FliD, and expression of PA flagellins in E. coli leads to fully formed and functional flagella. By comparison, Salmonella FliD does not mediate functional polymerization of Pseudomonas aeruginosa flagellins into flagella, and expression of Pseudomonas aeruginosa flagellins in Salmonella causes secretion into the cell supernatant (Inaba S et al., Biopolymers. 2013; 99(1):63-72). It has also been shown that Pseudomonas aeruginosa flagellin expressed in a heterologous GNB system is protective, as immunization with recombinant A-type flagellin produced in E. coli provided robust protection against burn wound infection with flagellin type A expressing Pseudomonas aeruginosa, including clinical isolates (Faezi S et al., APMIS. 2013). Monoclonal antibodies towards FlaA or FlaB, that have protected against burn wounds with the homologous Fla expressing Pseudomonas aeruginosa, recognize equivalently the cell-associated flagellin on Pseudomas aeruginosa and the recombinant soluble Pseudomas aeruginosa flagellin expressed in E. coli (Barnea Y et al., Burns. 2009; 35(3):390-396; Adawi A et al., Int J Mol Med. 2012; 30(3):455-464).
Immune responses towards the flagellins of several bacterial pathogens (e.g., Salmonella (Simon R, Infect Immun. 2011; 79(10):4240-4249; McSorley S J et al., J Immunol. 2000; 164(2):986-993), Pseudomas aeruginosa (Doring G et al., Vaccine. 2008; 26(8):1011-1024), Burkholderia (Brett P J et al., Infect Immun. 1996; 64(7):2824-2828)) have provided protection in animal models against infection. Flagellins have also been explored as carrier proteins for homologous pathogen bacterial surface polysaccharides. A conjugate vaccine comprised of Burkholderia pseudomallei COPS with the homologous strain flagellin (FliC) enhanced the anti-polysaccharide immune response, and antibodies induced by this vaccine imparted robust protection against B. pseudomallei infection (Brett P J et al., Infect Immun. 1996; 64(7):2824-2828). The inventors have found that conjugation of Salmonella entericaserovar Enteritidis COPS with S. Enteritidis flagellin enhances the anti-polysaccharide immune response and protects against fatal S. Enteritidis infection in mice (Simon R, Infect Immun. 2011; 79(10):4240-4249; Raphael Simon J Y W et al., PLOS ONE. 2013; 8(5):e64680). Conjugation of Pseudomas aeruginosa alginate polysaccharide with a recombinant A-type Pseudomas aeruginosa flagellin was also found to increase anti-alginate antibody levels, and elicit antibodies that protected by passive transfer against pneumonic PA infection with both mucoid and non-mucoid strains (Campodonico V L et al., Infect Immun. 2011; 79(8):3455-3464). Importantly, in all cases, antibody levels to polysaccharide conjugated flagellin were robust and equivalent to unconjugated flagellin, indicating that conjugation does not interfere with anti-flagellin immunity.
There remains a need for a broad spectrum vaccine that is effective against Klebsiella pneumoniae and Pseudomonas aeruginosa. The present invention provides multivalent conjugates directed against various Klebsiella pneumoniae serovars as well as Pseudomas aeruginosa for use in vaccines.
This background information is provided for informational purposes only. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the present invention.